Synthetic peptides, antibodies directed against them, and the use thereof

ABSTRACT

The invention relates to synthetic peptides which have amino acid sequences which correspond, in whole or in part, to the amino acid sequence of prothrombin and are antigenic, to the use thereof for the immunization of an animal and for the purification of specific antibodies, to antibodies against these peptides, and to the use of the antibodies for the determination of the fragments F 2 /F 1+2 . The antibodies hitherto used for the determination of the content of the fragments F 2 /F 1+2  have been induced by immunization with natural, highly purified prothrombin fragments F 2 /F 1+2 . The isolation of these prothrombin fragments is elaborate and costly, and the antibodies generated therewith show cross-reactions with intact prothrombin. Used for the immunization according to the invention are synthetic peptides which have amino acid sequences which correspond, in whole or in part, to the amino acid sequence of prothrombin and are antigenic. The resulting antibodies react specifically with the antigen used for the immunization; if the peptides contain the carboxyl-terminal amino acid sequence of the fragments F 2 /F 1+2  resulting after cleavage of the prothrombin molecule with factor Xa, then the antibodies generated react specifically with the fragments F 2 /F 1+2  but not with intact natural prothrombin, so that information can be gained about the degree of activation of prothrombin.

[0001] The invention relates to synthetic peptides, to the use of thesepeptides for immunizing an animal and for purifying specific antibodiesagainst the said peptides, to antibodies against these peptides, and tothe use of these antibodies.

[0002] The human organism has two systems, which are in equilibrium, toprotect itself both from blood loss and from thromboses: the coagulationsystem and the fibrinolytic system. The relationship between the twosystems ensures that insoluble fibrin polymers are produced initially tostop bleeding and, during wound healing, are cleaved again by the lyticprogress of fibrinolysis.

[0003] Plasmin and thrombin are the key enzymes in both systems. Underphysiological conditions, the dynamic equilibrium between thecoagulation and the fibrinolysis system is under the control of thethrombolytic activity of plasmin and the thromboplastic activity ofthrombin.

[0004] Thrombin is a typical serine protease and is synthesized in theform of an inactive precursor, prothrombin. Activation of prothrombin isbased on proteolysis by coagulation factor Xa, which represents acentral position within the coagulation cascade. Factor X itself has aspecial function in that it can be activated both by the extrinsic andby the intrinsic coagulation pathway. Activated factor X (factor Xa)activates prothrombin by specific cleavage of the prothrombin moleculeat the peptide bonds following the tetrapeptide Ile-Glu-Gly-Arg. Thiscleavage produces, on the one hand, thrombin and, on the other hand, inequimolar concentration the prothrombin fragment F₁₊₂. Since onemolecule of thrombin and one molecule of prothrombin fragment F₁₊₂ areproduced from each prothrombin molecule, cleaved, determination of theprothrombin fragment F₁₊₂ in blood or plasma allows a direct conclusionabout the coagulation potential, which depends on the thrombinconcentration present in blood or plasma.

[0005] The quantification of thrombin or the prothrombin fragmentsF₂/F₁₊₂ using radioimmunoassays is known from the state of the art. Theantisera required are produced by using the prothrombin fragments F₂ andF₁₊₂, obtained from purified prothrombin molecules for immunizinganimals. The specific antibodies are concentrated from the resultingcrude antisera by purification by immuno-adsorption on immobilizedprothrombin and the corresponding fragments. These antibody preparationsare suitable for the determination of the prothrombin fragments F₂/F₁₊₂,but do not enable a complete differentiation between intact uncleavedprothrombin and the prothrombin fragments F₂/F₁₊₂ liberated by cleavage.Furthermore, the relatively low specificity of this antibody preparationpermits determination of the antigen only by use of radioimmunoassays(RIA) which can be carried out in practice only if the conditions setout in the radiation protection regulations are observed and, for thisreason, require relatively great technical elaboration and financialcost. Finally, it is continually necessary to prepare fresh antibodieslabeled with radioactive isotopes because the isotope iodine-125normally used for the radiolabeling of proteins has a half-life of onlyabout 2 months.

[0006] Hence the object of the present invention was to provide antigenswhich result in the production of specific antibodies against theprothrombin fragments F₂/F₁₊₂ and thus allow rapid and accuratedetermination of the content of these fragments in biological fluids.

[0007] This object is achieved according to the invention by syntheticpeptides which have amino acid sequences which partly correspond to theamino acid sequence of prothrombin and are antigenic.

[0008] Thus the invention relates to peptides which have amino acidsequences which partly correspond to the carboxyl-terminal end of thefragments F₂/F₁₊₂ resulting from the FXa cleavage of thrombin, and whichcontain the amino acid sequenceH-Gly-Asp-Glu-Glu-Gly-Val-Trp-Cys-Tyr-Val-Ala-Gly-Lys-Pro-Gly-Asp-Phe-Gly-Tyr-Cys-Asp-Leu-Asn-Tyr-Cys-Glu-Glu-Ala-VaL-Gln-Glu-Glu-Thr-Gly-Asp-Gly-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH,in whole or in part, but at least the four carboxyl-terminal aminoacids.

[0009] Suitable for the preparation of the peptides according to theinvention are conventional methods, for example Merrifield solid-phasepeptide synthesis (G. Barany and R. B. Merrifield: “Solid-Phase PeptideSynthesis” in E. Gross and J. Meienhofer: The Peptides, Analysis,Synthesis, Biology, Adademic Press, Inc. 1980) as well as customarysynthetic strategies constructing the peptides in the form of solublepeptide segments. The peptide of the structureH-Cys(SH)-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg wasparticularly preferably prepared on the solid phase. The Fmoc group hasbeen used as temporary protective group, and the permanent protectivegroups used were the O-t-Bu protective group for Asp and Glu, t-Bu forSer, the Mtr group for Arg and the tert.-butylmercapto group for Cys.The C-terminal amino acids were immobilized via p-alkoxybenzyl estergroups which were bonded to 1% crosslinked polystyrene. The peptideswere constructed with repeated elimination of the temporary protectivegroup and condensation of the next, protected amino acid using acondensing agent, preferably a carbodiimide. The peptides were cleavedoff the resin by acidolysis with simultaneous deprotection of theside-chain groups. Any sulfhydryl groups to be deprotected aredeprotected using tri-n-butylphosphine according to the state of theart. The peptides were purified by ion exchange chromatography, RP-HPLCand gel permeation chromatography. The composition of the peptides wasconfirmed as correct by amino acid analysis.

[0010] The use of synthetic peptides as antigens in the immunization ofanimals results in the generation of antibodies specifically directedagainst the hapten exposed in this peptide. Hence the antibodiesgenerated in this way are each specific for a single antibody-bindingsite of the complete protein from which the peptide sequence has beenderived. Compared with the use of the natural, purified prothrombinfragments F₂/F₁₊₂, the use of synthetic peptides has two additionalprofound advantages; synthetic peptides can be prepared on a large scaleand in high purity so that the elaborate isolation and purification ofthe natural prothrombin fragments is avoided. Whereas the purificationof synthetic peptides from byproducts of the synthesis is wellestablished, even technically elaborate enrichment and purificationprocesses for natural prothrombin fragments always result inpreparations which contain a proportion of undesired peptides which,although not detectable still has antigenic activity.

[0011] The peptide prepared according to the invention has an amino acidsequence which corresponds completely or in part to the amino acidsequence of prothrombin and is synthesized by one of the conventionalprocesses for peptide synthesis, for example Merrifield synthesis. Theselection of the appropriate sequence entails, where possible, selectionof the regions which, due to their location on the protein and/or theantigenicity of the exposed epitope, can be predicted to have a highlyantigenic effect. The synthetic peptide then has antigenic activity, sothat an immune response is triggered by immunization.

[0012] Activated factor X (Xa) cleaves the prothrombin molecule at therecognition sequence Ile-Glu-Gly-Arg. Hitherto This tetrapeptide hasbeen detected only in human and bovine prothrombin. The rarity of thistetrapeptide predisposes it for use as a specific feature of theprothrombin molecule.

[0013] Prothrombin is cleaved by activated factor X next to the arginineof the sequence Ile-Glu-Gly-Arg. Thus, factor Xa generates a newcarboxyl terminus which contains in the terminal region the two aminoacids GLu and Arg which have a very high antigenicity index. Peptides orpolypeptides which contain the recognition sequence of the factor Xacleavage site at the C terminus are particularly well suited forimmunization.

[0014] It has emerged, surprisingly, that antibodies directed against apeptide or polypeptide which contains at its C-terminal end the sequenceof the tetrapeptide of the recognition sequence for the factor Xaprotease react specifically and exclusively with the prothrombinfragment which has been cleaved off and not with the intact prothrombinmolecule.

[0015] The fragment produced by the action of factor Xa on prothrombinand which has the factor Xa recognition sequence at the C terminus has achain length of 273 amino acids. Suitable for immunization are both theentire polypeptide as well as part-sequences of this peptide which,however, still need to have the factor Xa recognition sequence at the Cterminus. A particularly preferred embodiment provides for the use of atetradecapeptide, for example having the sequenceLeu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH. The onlyimportant point in each case is that the carboxyltenunal sequence of themolecule is exposed and results in the immunization.

[0016] Shorter peptides suffice in cases where it is not intended forthe peptide to trigger an immune response but it is intended that theonly function of this peptide is to be recognized by existingantibodies. An embodiment appropriate for this is based on anoctapeptide of the sequence Glu-Glu-Arg-ALa-lle-Glu-Gly-Arg-OH.

[0017] In view of the intended use of the peptides, it is appropriate tointroduce amino acids with reactive side groups into the peptides insuch a way that they do not affect the structure of the hapten. For thisreason, it is expedient to attach to the N terminus cysteine as afurther amino acid, the free SH group of which is suitable for couplingvia thioethers to many carriers. An example which is preferably providedis the antigen represented by the abovementioned peptide in the form ofthe pentadecapeptideCys(SH)-Leu-Asp-Glu-Asp-Ser-Asp-GLu-Glu-Arg-Ala-Ile-Glu-Gly-Arg.

[0018] The peptide used for the immunization can be prepared both bycustomary means by chemical synthesis and by purification of apolypeptide made available by genetic manipulation. A conceivableexample would be genetic engineering synthesis of the prothrombinfragment F₂ or F₁₊₂ in E. coli controlled by a strong promoter, or elsethe synthesis, by genetic manipulation, of a peptide extending beyondthe factor Xa cleavage site or a peptide which is subsequently cleavedin vitro by factor Xa, which is likewise obtained by geneticengineering, which results in accessibility to the desired antigeniccarboxyl terminus.

[0019] It is worthwhile for peptides which are intended to be used forimmunization, or those intended to be employed as an immunoadsorbent, tobe coupled to a carrier molecule. Examples of customary carriermolecules which are widely used are bovine serum albumin, ovalbumin andpolysaccharides. In a preferred embodiment, the peptide or polypeptideis bound to keyhole limpet hemocyanin.

[0020] When the synthetic peptides according to the invention are usedas immunoadsorbents, it is advisable to couple to materials suitable forpreparing solid matrices. A preferred embodiment provides for thecoupling of short peptides, for example the abovementioned octapeptide,to Sepharose which has been activated with cyanogen bromide.

[0021] Immunization of suitable animals with carrier-bound peptidesresults reproducibly in the production of antibodies. A preferred animalspecies for immunization and obtaining antibodies in this context is therabbit, because in this case there is a favorable relationship betweenthe blood volume obtainable and the expense of breeding and care.

[0022] The immunoglobulin fraction relevant for specific assays can beenriched by customary immunoadsorption methods from an antiserum of thistype generated in an animal using synthetic peptides according to theinvention. However, it is preferred in this case likewise to use asmaterial for a matrix of this type, which is employed forimmunoadsorption, a peptide which is coupled to a carrier and has thesame antigenic determinant as the peptide employed for the immunization.The peptide used for the purification by immunoadsorption can moreoverbe considerably shorter; the only prerequisite for use in purificationof the desired antibody by immunoadsorption is that the antigenicdeterminant formed by this shorter polypeptide is recognized andefficiently bound by the desired antibody.

[0023] The peptide used for purification of the antibodies byimmunoadsorption can be, for example, an octapeptide, preferably thepeptide H-Glu-Glu-Arg-Ala-Ile-Glu-GLy-Arg.

[0024] According to the invention, antibodies are induced in the animalsystem by immunization with synthetic peptides and are purified byimmunoadsorption. These antibodies react specifically with the peptidesused for the immunization and purification. Depending on the sequence ofthe peptide used, these antibodies bind either only to the fragments F₂and F₁₊₂ or else, if a peptide sequence which is exposed in the naturalprothrombin molecule is chosen, to the intact prothrombin molecule.

[0025] It is possible, by choosing appropriate peptides asimmunoadsorbents, to select antibodies which react specifically with theantigenic determinant of prothrombin which corresponds to the sequenceof the factor Xa cleavage site of this molecule; in the preferred casewhere peptides which have the factor Xa-recognition sequence at the Cterminus are used both for the immunization and for the purification byimmunoadsorption there is enrichment of antibodies against thissequence, but these do not react with intact natural prothrombin,because the factor Xa cleavage site in the natural prothrombin moleculeeither is not sufficiently exposed or does not have the higher structurerequired for recognition as an antigen.

[0026] The antibodies obtained according to the invention can beemployed for a number of immunoassays with a variety of designs. Forthis purpose, they are expediently coupled to a solid carrier, but arepreferably immobilized by adsorption onto polystyrene tubes. The tubesprepared for the following immunoassays can then be stored, closedairtight, at 4° C.

[0027] The amount of prothrombin fragments F₂/F₁₊₂ is determinedaccording to the invention by preincubation of the sample withimmobilized antibodies of this type, with the concentration of thefragments F₂/F₁₊₂ which are bound by the immobilized antibodies beingdetected by subsequent incubation with a second antibody. This secondantibody must have a property which is measurable, for example theability to react with or bind a chromogenic substrate.

[0028] It is expedient for this second antibody to be coupled to amarker enzyme, preferably peroxidase. However, it is also possible tochoose to provide the second antibody either with a fluorescentmolecule, for example fluorescein isothiocyanate or else with aradioactive label.

[0029] The prothrombin fragments F₂/F₁₊₂ can also be determined bysimultaneous incubation of the sample, preferably of plasma, and labeledantibodies with the immobilized antibodies. Also possible is acompetitive determination method entailing competition of labeled andunlabeled prothrombin fragments F₂/F₁₊₂ for the binding site on theimmobilized antibodies.

[0030] The concentration of prothrombin fragments F₂/F₁₊₂ determined inthis way provides information on the degree of activation of theprothrombin.

[0031] The examples illustrate the invention. The followingabbreviations are used in the examples: FPA fibrinopeptide A ELISAenzyme immunoassay (enzyme-linked immunosorbent immunosorbent assay) RIAraddio immunoassay KLH keyhole limpet hemocyanin PBS phosphate-bufferedsaline Tris tris(hydroxymethyl )aminomethane EDTAethylenedinitrilotetraacetic acid OD absorbance (optical density) AspL-aspartic acid Ala L-alanine Arg L-arginine Gly glycine Glu L-glutamicacid Ile L-isoleucine Ser L-Serine Cys(SH) L-cysteine Fmoc9-fluorenylmethytoxycarbonyl O-t-Bu tert.-butyl ester t-Bu tert.-butylether Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl DMF dimethyl formamideRP-HPLC reversed phase high performance Liquid chromatography GMBSgamma-maleimidobutyric acid hydroxysuccinimide ester

EXAMPLE 1

[0032] Preparation Of an Antigen For the Immunization

[0033] a) Peptide Synthesis For the PentadecapeptideH-Cys(SH)-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg

[0034] The peptide was synthesized using a semiautomatic peptidesynthesizer. 1 g of Fmoc-Arg (Mtr)-p-alkoxybenzyl ester-resin wasdeprotected with 15 ml of 20% piperidine/DMF (v/v) and then washedseveral times with DMF and isopropanol. 1 mmol of Fmoc-Gly (3-foldexcess) and 203 mg of HOBt dissolved in 15 ml of DMF were added. 1.1 mlof a 1 M diisopropylcarbodiimide solution (dichloromethane) were addedand then the coupling was carried out for 1.5 hours. Excess reagentswere removed by washing with DMF and isopropanol. This coupling schemewas maintained up to the N-terminal amino acid. The last amino acidemployed was a Boc-protected amino acid. Every coupling step was checkedfor completeness by a ninhydrin test. 1.06 g of resin were stirred with2.5 ml of thioanisole, 2.5 ml of ethanedithiol and 15 ml oftrifluoroacetic acid at 35° C. for 4 hours and were filtered off. Theacidic solution was poured into ether, and the precipitated peptide wasfiltered off and chromatographed on a Sephadex^((R)) G 25 column (3×100cm, 0.5% acetic acid). The peptide pool was lyophilized. Yield: 230 mgof peptide.

[0035] b) Deprotection Of the Sulfhydryl Group

[0036] 70 mg of the peptide were dissolved in 7 ml of trifluoroethanoland 350 μl of water, and the pH was adjusted to 7.3 withN-methylmorpholine. The reaction vessel was flushed with nitrogen, and40 μl of n-tributylphosphine were added. The mixture was stirred at roomtemperature for 1 hour and was diluted with 50 ml of water, and the pHwas adjusted to 4.0. The aqueous phase was extracted three times with 10ml of diethyl ether, concentrated to 10 ml and purified onSephadex^((R)) G 25 (3×100 cm; 0.5% acetic acid). Yield: 55 mg ofpeptide.

[0037] c) Preparation Of Conjugate

[0038] 30 mg of keyhole limpet hemocyanin were dissolved in 0.05 mMsodium phosphate buffer, pH 8.0, and were activated with 3 mg of GMBSfor 1 hour. The protein was chromatographed on a Sephadex^((R)) G 50column (2×30 cm) (0.1 M sodium phosphate; 0.5 mM EDTA, pH 6.0). Theprotein pool was concentrated to 6 ml and incubated with 30 mg of thepeptide containing sulfhydryl groups for 1 hour. Yield after dialysisand lyophilization: 38 mg of peptide conjugate.

Example 2

[0039] Immunization Of Rabbits

[0040] 5 rabbits were immunized with 2 mg of antigen per animal eachtime for a period of 8 weeks; the peptide-KLH conjugate administrationswere given subcutaneously and intravenously. The animals were thenexsanguinated, and the resulting crude antisera were pooled andstabilized with preservative. Yield: 850 ml of antiserum.

Example 3

[0041] Preparation Of Immunoadsorbents

[0042] For the purification of the crude antisera by affinitychromatography, about 20 mg of the octapeptide with the sequenceH-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH (prepared as in Example 1a) werecovalently immobilized on a solid phase. The coupling reaction wascarried out with Sepharose activated with cyanogen bromide by adescribed method (Axen, R. et al., Nature, 214, 1302, 1967). Theimmunoadsorbent was then in each case washed with phosphate-bufferedsaline (PBS; 0.15 mol/l, pH 7.2) and acetic acid (0.5 mol/l, pH 2.5).Before use, the adsorbent was equilibrated with 3 times the gel volumeof PBS. Yield: about 20 ml of peptide-Sepharose.

[0043] The tetradecapeptide prepared as in Example 1 was used in thesame way to prepare an immunoadsorbent.

Example 4

[0044] Isolation Of Specific Antibodies

[0045] 100 ml of crude antiserum were applied to theoctapeptide-Sepharose (1.5×15 cm) equilibrated with PBS, andsubsequently washed with PBS until the absorbance at 280 nm was 0.01.This was followed by washing steps with saline (1 mol/l, pH 7.0) andwater (pH 7.0), with 3 times the gel volume being used in each case. Theantibodies were eluted from the immunoadsorbent with 0.1 mol/l aceticacid (pH 2.5), and the antibody solution was adjusted to pH 7.0 withsolid sodium phosphate (0.01 mol/l), concentrated (Amicon membrane) andstored at −70° C. Yield: 35 to 40 mg of antibody.

Example 5

[0046] Assays Of the Antibodies Obtained By Immunoadsorption.

[0047] a) Preparation Of Antibody-Coated Tubes

[0048] The antibodies obtained in Example 4 were diluted with trisbuffer solution (0.025 mol/l, pH 7.6) to a concentration of 5 μg/ml andimmobilized by adsorption onto polystyrene tubes. 250 μl of antibodysolution were incubated in each tube at 20° C. for 20 hours, and thenthe liquid was removed by aspiration, and the tubes were sealed airtightand stored at 4° C.

[0049] b) Procedure For the Enzyme Immunoassay (ELISA)

[0050] The samples to be assayed (plasma, serum) were diluted 1:1 withincubation buffer (0.01 mol/l tris, 0.01 mol/L EDTA, heparin (2 U/mL),0.05% Tween, pH 7.6), and 200 μl samples in each tube (see Example 5a)were incubated at 37° C. for 30 min. The incubation solution was thenremoved, and the tube was washed twice with 500 μl of washing solutioneach time (0.02 mol/l sodium phosphate, 0.05% Tween, pH 7.6). Then 200μl of peroxidase-conjugated anti-prothrombin antibodies were added, andthe tubes were incubated at 37° C. for 30 min. After removal of theconjugate solution and two washes, 200 μl of substrate/chromogensolution (hydrogen peroxide; o-phenylenediamine) were added, and thetubes were incubated at room temperature. After incubation for ½ anhour, the peroxidase was inactivated with sulfuric acid, and theabsorbance of the reaction solution at 492 nm was determined.

[0051] The absorbances at 492 nm as a function of the plasma or serumdilution are shown in the table which follows, comparing with theabsorbance of a tube without plasma or serum. TABLE 1 Dilution OD₄₉₂/30min Plasma 1:10 0.13 1:100 0.12 1:1000 0.11 1:10000 0.11 Serum 1:10 3.721:100 3.71 1:1000 2,81 1:10000 0.97 Guffer blank 0.045

[0052] c) Procedure For an Enzyme Immunoassay (ELISA) For DeterminingFragments F₂/F₁₊₂ Formed In Vitro.

[0053] Another experiment was carried out to examine the specificity ofthe antibodies against the fragments F₂/F₁₊₂. Plasma anticoagulated withcitrate solution was recalcified with calcium chloride solution (finalconcentration of calcium chloride: 0.025 mol/l). Aliquots were removedat various times, and the reaction was stopped by addition of EDTA (0.1mol/l), antithrombin III (3 IU/mL) and heparin (5 IU/ml). The sampleswere diluted 1:1 with incubation buffer and assayed using the ELISA.

[0054] The table shows the results. TABLE 2 Time (min) OD₄₉₂/39 min.  00, 18  3 0, 30  6 0, 32 12 0, 58 15 1, 20 18 1, 69 21 1, 88 25 1, 96Buffer blank 0, 045

[0055] The results show that the fragments F₂/F₁₊₂ can be measuredquantitatively in this way: the concentration of the fragments F₂/F₁₊₂increases with increasing time during the recalcification reaction.

[0056] The peptides according to the invention, which have an amino acidsequence which corresponds, in whole or in part, to the amino acidsequence of prothrombin and is antigenic thus induce binding-specificantibodies against each of the antigenic determinants present in thepeptide. These specific antibodies can then be purified byimmunoadsorption on peptides having the same antigenic determinants. Theuse of synthetic peptides has the considerable advantage that absolutelypure antigens are employed for the immunization, so that nocross-reaction whatever with other proteins or other parts of theprothrombin molecule can occur in the resulting immune serum. Preferablyused according to the invention is a peptide which contains the factorXa cleavage site. Using an antibody against this peptide it is possibleto detect only cleaved prothrombin molecules, because this sequence isnot accessible to antibody recognition in intact natural prothrombin.Measurement of the quantity of bound antibody provides directinformation on the concentration of liberated prothrombin fragmentsF₂/F₁₊₂ and thus on the degree of activation of prothrombin.

1 5 1 4 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 1 Ile Glu Gly Arg 1 2 50 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 2 Gly Asp Glu GluGly Val Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp 1 5 10 15 Phe Gly TyrCys Asp Leu Asn Tyr Cys Glu Glu Ala Val Gln Glu Glu 20 25 30 Thr Gly AspGly Leu Asp Glu Asp Ser Asp Glu Glu Arg Ala Ile Glu 35 40 45 Gly Arg 503 15 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 3 Cys Leu Asp Glu Asp Ser Asp Glu Glu Arg Ala Ile GluGly Arg 1 5 10 15 4 14 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 4 Leu Asp Glu Asp Ser Asp Glu Glu Arg Ala IleGlu Gly Arg 1 5 10 5 8 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 5 Glu Glu Arg Ala Ile Glu Gly Arg 1 5

1. Peptides which have amino acid sequences which partly correspond tothe carboxyl-terminal end of the fragments F₂/F₁₊₂ resulting from the FXa cleavage of prothrombin, and which contain the amino acid sequenceH-Gly-Asp-Glu-Glu-Gly-Val-Trp-Cys-Tyr-Val-Ala-Gly-Lys-Pro-Gly-Asp-Phe-Gly-Tyr-Cys-Asp-Leu-Asn-Tyr-Cys-Glu-Glu-Ala-Val-Gln-Glu-Glu-Thr-Gly-Asp-Gly-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH,in whole or in part, but at least the four carboxyl-terminal aminoacids.
 2. A peptide as claimed in claim 1, which has the amino acidsequence H-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-ILe-Glu-Gly-Arg-OH,H-Glu-GLu-Arg-Ala-ILe-Glu-Gly-Arg-OH orH-Cys(SH)-Leu-Asp-GLu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg. 3.Peptides as claimed in claim 1, which are prepared by geneticmanipulation or by chemical synthesis.
 4. A peptide as claimed in claim1, which is bound to an insoluble polymeric carrier or keyhole limpethemocyanin, albumin or ovalbumin.
 5. The use of the peptides as claimedin claim 1 for the immunization of an animal, preferably of a rabbit,and for the purification of specific antibodies against the said peptideby means of immunoadsorption.
 6. The use of a polypeptide as claimed inclaim 5, wherein the peptide has the amino acid sequenceH-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH.
 7. Antibodies against peptides asclaimed in claim 1, which have been obtained by immunization of ananimal with a peptide as claimed in at least one of claims 1 to 4 andsubsequent purification by immunoadsorption as claimed in at least oneof claims 5 or
 6. 8. Antibodies as claimed in claim 7, which reactspecfically with a peptide as claimed in claim 1 and/or with naturalprothrombin or parts thereof.
 9. Antibodies as claimed in claim 8, whichare specific for binding to the prothrombin fragments F₂/F₁₊₂ and arecoupled to a carrier.
 10. A method for the determination of prothrombinfragment F₁₊₂/F₂ using antibodies as claimed in at least one of claims 7to 9, which comprises the prothrombin fragments F₂/F₁₊₂ being bound tothe specific antibodies and being detected by a second antibody, andcomprises the quantity of the bound second antibody being measurable.11. The use of antibodies as claimed in claim 10, wherein a markerenzyme, preferably peroxidase, is bound to the second antibody.